Single-cell transcriptomic analysis reveals alterations to cellular dynamics and paracrine signalling in radiation-induced muscle pathology

Radiation therapy causes long-term skeletal muscle atrophy and fibrosis in juvenile cancer survivors. The mechanisms responsible for the skeletal muscle late effects of radiation therapy are not well-understood and have prevented the development of effective treatments. Using single-cell RNA sequencing (scRNA-seq), we characterize cellular dynamics and communication in a murine model of therapeutic radiation at 24-hours and 56-days post-irradiation (post-IR). We detected changes in muscle stem (satellite) cells (MuSCs) characterized by an acute preservation of committed MuSCs and long-term relative depletion of deep quiescent MuSCs. A conserved senescence Cdkn1a signature was observed in all muscle-resident cells post-IR. Genes related to fibroblast proliferation were up-regulated and a fibrotic and senescent transcriptome persisted in Fibro-adipogenic progenitors (FAPs) post-IR. Intercellular communication analysis revealed FAPs as the primary contributor of extracellular matrix (ECM) and target of monocyte/macrophage-derived TGF-β signalling post-IR through TGF-βR2 on FAPs. Together, our findings provide insights into the potential mechanisms and intercellular communication responsible for radiation-induced muscle atrophy and fibrosis. A fractionated dose of in vivo irradiation (in vivo-IR) of 8.2 Gy/dose was administered for three days to one hindlimb at age five weeks (IR). The rest of the body was lead-shielded, and the contralateral limb was used as a non-IR control (CON). Three independent samples (IR or CON limb) were pooled together in one batch to avoid batch effects. IR or the CON limb was removed at 24-hours post the final dose of radiation or 56-days post-IR (CON 24-hours, IR 24-hours, CON 56-days, and IR 56-days). A minimum of 10,000 live, mononucleated cells were sorted by Fluorescence-activated cell sorting (FACS) for further scRNA-seq (10X Genomics, Chromium Single Cell 30 Reagent Kits (v3 Chemistry)). The four libraries (CON 24-hours, IR 24-hours, CON 56-days, and IR 56-days) were sequenced on the Illumina NextSeq 500, with a read depth of 10,000 reads per cell.